U.S. patent number 7,618,587 [Application Number 10/629,296] was granted by the patent office on 2009-11-17 for analyzers and methods of analyzing blood.
This patent grant is currently assigned to Sysmex Corporation. Invention is credited to Yasunori Kawate.
United States Patent |
7,618,587 |
Kawate |
November 17, 2009 |
**Please see images for:
( Certificate of Correction ) ** |
Analyzers and methods of analyzing blood
Abstract
Analyzers and methods of analysis are described for performing
blood cell counting and immunoassay on a whole blood specimen in
one measurement section. An assay sample is prepared by blending
carrier particles sensitized with an antibody or an antigen against
a substance to be immunoassayed and a fluorescent dye for staining
blood cells with the whole blood specimen. Optical information is
detected from a particle in the assay sample, and the blood cells
are differentiated and counted based on the detected optical
information. A rate of agglutination of the carrier particles is
obtained based on the detected optical information, thereby
enabling detection of the substance to be immunoassayed.
Inventors: |
Kawate; Yasunori (Kakogawa,
JP) |
Assignee: |
Sysmex Corporation (Kobe-shi,
JP)
|
Family
ID: |
30117488 |
Appl.
No.: |
10/629,296 |
Filed: |
July 28, 2003 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20040018629 A1 |
Jan 29, 2004 |
|
Foreign Application Priority Data
|
|
|
|
|
Jul 29, 2002 [JP] |
|
|
2002-219187 |
Jun 30, 2003 [JP] |
|
|
2003-188895 |
|
Current U.S.
Class: |
422/73; 422/119;
422/50; 422/504; 422/67; 422/68.1; 422/82.05; 422/82.08; 422/82.09;
435/287.2; 435/7.1; 435/7.21; 435/7.25; 436/10; 436/164; 436/165;
436/172; 436/518; 436/523; 436/524; 436/528 |
Current CPC
Class: |
G01N
15/1468 (20130101); G01N 33/54313 (20130101); Y10T
436/101666 (20150115); G01N 2015/1486 (20130101) |
Current International
Class: |
G01N
21/66 (20060101) |
Field of
Search: |
;422/50,55,67,73,68.1,82.05,99,108,119,105,187-189,82.08,82.09
;435/7.1,7.2,7.21,7.25,7.8,287.2,962
;436/63,164,172,523-529,531-534,514,536,538,10,16,165,175,518 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
0 347 210 |
|
Dec 1989 |
|
EP |
|
0 455 125 |
|
Nov 1991 |
|
EP |
|
0 722 087 |
|
Jul 1996 |
|
EP |
|
B-19349 |
|
Nov 1983 |
|
JP |
|
H6-19349 |
|
Mar 1994 |
|
JP |
|
11-101798 |
|
Apr 1999 |
|
JP |
|
WO 92/09682 |
|
Jun 1992 |
|
WO |
|
WO 02/23154 |
|
Mar 2002 |
|
WO |
|
Other References
Ruzicka, MD, K.; Veitl, MD, M.; Thalhammer-Scherrer, MD, R.;
Schwarzinger, MD, I., "The New Hematology Analyzer Sysmex XE-2100:
Performance Evaluation of a Novel White Blood Cell Differential
Technology", Arch Pathol Lab Med, Mar. 2001, vol. 125, pp. 391-396.
cited by other.
|
Primary Examiner: Gabel; Gailene R
Attorney, Agent or Firm: Brinks Hofer Gilson & Lione
Claims
What is claimed is:
1. An analyzer comprising: a sample preparing portion configured
for preparing an assay sample comprising a reagent and a whole
blood specimen, the sample preparing portion comprising: a reaction
vessel and a reagent supplying portion for supplying the reagent to
the reaction vessel, wherein the reagent comprises fluorescent
carrier particles sensitized with an antibody or an antigen against
a target substance found in the serum or blood plasma portion of
the whole blood specimen; a flow cell; an assay sample supplier for
supplying the assay sample from the reaction vessel to the flow
cell; a light source for irradiating the assay sample in the flow
cell; a first detector that detects fluorescence intensities from
irradiated particle components including blood cells and
fluorescent carrier particles that reacted to the target substance
in the assay sample; a second detector that detects scattered light
intensities from irradiated particle components including blood
cells and fluorescent carrier particles that reacted to the target
substance in the assay sample; and an analyzing portion device
configured: differentiate the blood cells and the fluorescent
carrier particles based on the detected fluorescence intensities
received from the first detector and the detected scattered light
intensities received from the second detector; count the
differentiated blood cells; and differentially detect agglutination
degree of the fluorescent carrier particles that reacted to the
target substance.
2. The blood analyzer of claim 1, further comprising a second
reagent supplying portion for supplying the reagent to the reaction
vessel, wherein the assay sample further comprises a second reagent
comprising a fluorescent dye for staining blood cells.
3. The blood analyzer of claim 2 wherein the analyzing portion
differentiates blood cells into erythrocytes, leukocytes, and
platelets, and wherein the analyzing portion counts the
differentiated blood cells.
4. The blood analyzer of claim 1, wherein the operations further
comprise: obtaining a concentration value of the target substance
based on the detected agglutination degree; and correcting the
concentration value as a whole blood immunoassay result to a
concentration value as a serum or plasma immunoassay result based
on a result of blood cell counting.
5. The blood analyzer of claim 1, wherein the operations further
comprise: obtaining a concentration value of the target substance
based on the detected agglutination degree; obtaining a hematocrit
value based on size information of blood cells; and correcting the
concentration value as a whole blood immunoassay result to a
concentration value as a serum or plasma immunoassay result based
on the hematocrit value.
6. An analyzer comprising: a sample preparing portion configured
for preparing an immunoassay sample for an immunoassay by adding a
first reagent for the immunoassay to a first specimen split from a
whole blood specimen, and for preparing a counting sample for blood
cell counting by adding a second reagent for the blood cell
counting to a second specimen split from the whole blood specimen;
wherein the first reagent comprises fluorescent carrier particles
sensitized with an antibody or an antigen against a target
substance found in the serum or blood plasma portion of the whole
blood specimen; wherein the second reagent comprises a fluorescent
dye for staining blood cells; and wherein the sample preparing
portion comprises a first reaction vessel for preparing the
immunoassay sample, a second reaction vessel for preparing the
counting sample, a first reagent supplying portion for supplying
the first reagent to the first reaction vessel, and a second
reagent supplying portion for supplying the second reagent to the
second reaction vessel; a flow cell; an immunoassay sample supplier
for supplying the immunoassay sample from the first reaction vessel
to the flow cell; a counting sample supplier for supplying the
counting sample from the second reaction vessel to the flow cell; a
light source for irradiating the immunoassay sample or the counting
sample in the flow cell; a first detector that detects fluorescence
intensities from irradiated particle components including
fluorescent carrier particles that reacted to the target substance
contained in each of the immunoassay sample and the counting
sample; a second detector that detects scattered light intensities
from irradiated particle components including fluorescent carrier
particles that reacted to the target substance contained in each of
the immunoassay sample and the counting sample; and an analyzing
portion device configured to: differentiate the fluorescent carrier
particles from the blood cells based on the detected fluorescence
intensities of the immunoassay sample by the first detector and the
detected scattered light intensities transmitted by the second
detector; differentially detect agglutination degree of the
fluorescent carrier particles that reacted to the target substance;
differentiate the blood cells based on the detected fluorescence
intensities of the count sample transmitted by the first detector
and the detected scattered light intensities of the counting sample
transmitted by the second detector; and counting the differentiated
blood cells.
7. The blood analyzer of claim 6, wherein the analyzing portion
differentiates blood cells into erythrocytes, leukocytes, and
platelets, and wherein the analyzing portion counts the
differentiated blood cells.
8. The blood analyzer of claim 6, wherein the operations further
comprise: obtaining a concentration value of the target substance
based on the detected agglutination degree; and correcting the
concentration value as a whole blood immunoassay result to a
concentration value as a serum or plasma immunoassay result based
on a result of blood cell counting.
9. The blood analyzer of claim 6, wherein the operations further
comprise: obtaining a concentration value of the target substance
based on the detected agglutination degree; obtaining a hematocrit
value based on size information of blood cells; and correcting the
concentration value as the whole blood immunoassay result to a
concentration value as a serum or plasma immunoassay result based
on the hematocrit value.
10. An analyzer comprising: a sample preparing portion configured
for preparing an assay sample comprising a reagent and a whole
blood specimen, the sample preparing portion comprising: a reaction
vessel and a reagent supplying portion for supplying the reagent to
the reaction vessel, wherein the reagent comprises fluorescent
carrier particles sensitized with an antibody or an antigen against
a target substance found in the serum or blood plasma portion of
the whole blood specimen; a flow cell; an assay sample supplier for
supplying the assay sample from the reaction vessel to the flow
cell; a light source for irradiating the assay sample in the flow
cell; a first detector that detects fluorescence intensities from
irradiated particle components including blood cells and
fluorescent carrier particles that reacted to the target substance
in the assay sample through a first device that converts a first
non-electrical energy into first electrical energy; a second
detector that detects scattered light intensities from irradiated
particle components including blood cells and fluorescent carrier
particles that reacted to the target substance in the assay sample
through a second device that converts a second non-electrical
energy into second electrical energy; and an analyzing system
comprising: a differentiating processor that differentiates blood
cells and the fluorescent carrier particles that reacted to the
target substance by processing output transmitted by the first
detector and the second detector; a measuring detector that counts
the differentiated blood cells; and an agglutination processor in
communication with the second detector that differentially detects
agglutination degree of the fluorescent carrier particles that
reacted to the target substance.
11. The analyzer of claim 10 further comprising a visual output
device in communication with the agglutination processor to display
physical agglutination patterns.
12. The analyzer of claim 10 where the agglutination processor
comprises means for detecting agglutination degree of the
fluorescence carrier particles based on the detected scattered
light intensities captured in the output of the second detector.
Description
RELATED APPLICATIONS
This application claims priority under 35 U.S.C. .sctn. 119 to
Japanese Patent Application Nos. 2002-219187, filed Jul. 29, 2002,
and 2003-188895, filed Jun. 30, 2003.
FIELD OF THE INVENTION
The present invention relates to blood analyzers and analysis
methods for efficiently performing blood cell counting and
immunoassay on a specimen.
BACKGROUND
In the field of laboratory tests, various analysis apparatuses such
as blood cell counters, immunoassay apparatuses, blood coagulation
analyzers, and biochemical analyzers are used depending on the
desired measurement parameters. Thus, it is typically necessary to
manage multiple analysis apparatuses. Moreover, it is typically
necessary to collect multiple specimens from a patient depending on
the parameters to be measured, which places a considerable burden
on the patient. Consequently, an automated analyzer capable of
analyzing multiple parameters in a single specimen would be
desirable.
Blood cell counting involves differentiating blood cells contained
in blood (i.e., whole blood) and counting according to blood cell
type. The blood cells are generally differentiated into
erythrocytes, leukocytes, platelets, and the like. Thus,
erythrocyte number, leukocyte number, and platelet number are
representative parameters of blood cell counting. In addition,
reticulocytes which emerge in peripheral blood in an immature state
of erythrocytes are differentiated and counted in some cases.
An analysis apparatus for blood cell counting includes an automated
hematology analyzer, such as the XE-2100 supplied by Sysmex
Corporation. Here, blood cells are stained with specific
fluorescent dyes, optical information (e.g., forward scattered
light, side scattered light and fluorescence) is detected from the
respective blood cells by flow cytometry, and the blood cells are
differentiated and counted by combining this optical information.
In addition, this analysis apparatus has a counting function for
reticulocytes, whereby forward scattered light intensity and side
fluorescence intensity are detected from the fluorescently stained
blood cells by reacting with staining solution without hemolysis.
Two dimensional scattergrams are made using these as parameters to
differentiate the blood cells into platelets, erythrocytes,
reticulocytes and the like. The staining solution for fluorescent
staining of the blood cells contains a dye which stains nucleic
acid contained in the blood cells, and stains the leukocytes and
reticulocytes. The side fluorescence intensity detected from the
blood cells provides information indicative of the amount of
nucleic acid in the blood cells, and the blood cells can be
differentiated by combining the forward scattered light intensity
(size information) and the side fluorescence intensity (nucleic
acid amount information).
In addition to blood cell number, mean corpuscular volume (MCV) and
hematocrit value are also used as parameters of blood cell
counting. MCV is a mean value of erythrocyte sizes in whole blood.
The hematocrit value is a percentage of blood cell component
occupying the whole blood. Since an erythrocyte volume occupies a
vast majority of the blood cell volume, the hematocrit value is
calculated by measuring the erythrocyte number and MCV in the whole
blood, multiplying the MCV by the erythrocyte number in the whole
blood, and dividing it by the volume of the whole blood.
An immunoassay is an assay method for making an antigen or an
antibody contained in a specimen (e.g., blood) a substance to be
assayed, which is detected by taking advantage of an antigen
antibody reaction. Representative immunoassays include an enzyme
immunoassay (EIA) method, a radioimmunoassay (RIA) method, a
particle agglutination method, and the like. The particle
agglutination method is a method in which a substance to be
immunoassayed is detected by blending carrier particles sensitized
with an antibody or an antigen corresponding to the substance to be
assayed with a sample, inducing a particle agglutination reaction
due to the antigen antibody reaction, and measuring the degree of
the particle agglutination (degree of agglutination) from changes
in absorbance and light scatter.
In conventional particle agglutination methods, a sample containing
carrier particles after the agglutination reaction is measured by
flow cytometry and the degree of agglutination is obtained based on
optical information obtained from the respective particles. When
the information which reflects size of the carrier particles (e.g.,
forward scattered light) is used as the optical information,
unagglutinated carrier particles can be discriminated from
agglutinated carrier particles, and the degree of agglutination of
the carrier particles can be obtained. A rate of agglutination
method for determining degree of agglutination is described in
JP-B-6-19349. In this method, scattered light intensities of
respective particles are measured by a flow cytometer.
Non-agglutinated single particles and agglutinated particles which
occur by agglutinating multiple carrier particles are
differentiated according to their respective scattered light
intensities. Single particle number (M) and agglutinated particle
number (P) are counted to obtain a total particle number (T) which
is a sum of M and P, and P/T is calculated as the rate of
agglutination. Since the reaction can be caught at a stage where
two carrier particles are agglutinated, an extremely high
sensitivity immunoassay becomes possible. In this rate of
agglutination assay, various methods (e.g., in which the rate of
agglutinated particles measured equals or exceeds a certain number)
can be used depending on the assay level range of the substance to
be immunoassayed. The rate of agglutination assay method in the
above-described JP-B-6-19349 is used for the immunoagglutination
assay apparatus PAMIA series supplied by Sysmex Corporation.
Whole blood, serum, plasma, and the like are used as samples in the
above-described apparatuses for blood cell counting and
immunoassay. However, while whole blood samples are typically used
in the blood cell counter, serum or plasma are typically used in
other apparatuses (e.g., the immunoassay apparatuses). A blood cell
counting/immunoassay apparatus using whole blood in which blood
cell counting and immunoassay can both be carried out is described
in U.S. Pat. No. 6,106,778. This apparatus has a blood cell
counting portion and an immunoassay portion, and measures by
dispensing the whole blood sample into the blood cell counting
portion and the immunoassay portion, respectively. In the
immunoassay portion, the immunoassay is carried out by hemolysing
the whole blood sample with a hemolytic agent and using latex
reagents.
However, when both blood cell counting and immunoassay are to be
performed, it would be highly desirable that the blood cell
counting and the immunoassay be carried out in an identical
measurement section in order to reduce the amount of specimen
collected from a patient and to enable measurement by a single
small analyzer.
SUMMARY OF THE INVENTION
The scope of the present invention is defined solely by the
appended claims, and is not affected to any degree by the
statements within this summary.
A first analyzer embodying features of the present invention
includes (a) a sample preparing portion configured for preparing an
assay sample, wherein the assay sample contains at least one
reagent and a blood specimen; (b) a light source for irradiating
the assay sample; (c) a light detector for detecting optical
information from a particle in the assay sample; and (d) an
analyzing portion where blood cell counting and detection of a
substance to be immunoassayed are carried out based on the optical
information detected by the light detector.
A second analyzer embodying features of the present invention
includes (a) a sample preparing portion, which is configured for
preparing a sample for an immunoassay by adding a reagent for the
immunoassay to one of at least two split blood specimens, and for
preparing a sample for blood cell counting by adding a reagent for
the blood cell counting to another of the at least two split blood
specimens; (b) a light source for irradiating the sample for
immunoassay and the sample for blood cell counting; (c) a light
detector for detecting optical information from a particle in each
of the sample for immunoassay and the sample for blood cell
counting; and (d) an analyzing portion, wherein a substance to be
immunoassayed is detected based on the optical information detected
from the particle in the sample for immunoassay, and wherein the
blood cell counting is performed based on the optical information
detected from the particle in the sample for blood cell
counting.
A third analyzer embodying features of the present invention
includes (a) a sample preparing portion configured for preparing an
assay sample by blending carrier particles sensitized with an
antibody or an antigen against a substance to be immunoassayed and
a fluorescent dye for staining blood cells with a blood specimen;
(b) a light detecting portion containing a flow cell for flowing
the assay sample, a light source for irradiating the assay sample
flowing through the flow cell, and a detector for detecting forward
scattered light and fluorescence emitted from a particle in the
assay sample; and (c) an analyzing portion, wherein blood cell
count and detection of the substance to be immunoassayed are
performed based on the forward scattered light and the fluorescence
detected by the light detecting portion.
A fourth analyzer embodying features of the present invention
includes (a) a sample preparing portion configured for preparing an
assay sample by adding at least one reagent to a blood specimen; a
detecting portion for detecting a physical property of a particle
in the assay sample; and an analyzing portion for performing blood
cell counting and detection of a substance to be immunoassayed
based on the physical property detected by the detecting
portion.
A first method of analyzing blood embodying features of the present
invention includes (a) preparing an assay sample by adding at least
one reagent to a blood specimen; (b) irradiating the assay sample;
(c) detecting optical information from a particle in the assay
sample; and (d) performing blood cell counting and detection of a
substance to be immunoassayed based on the optical information
detected.
A second method of analyzing blood embodying features of the
present invention includes (a) preparing a sample for an
immunoassay by adding a reagent for the immunoassay to one of at
least two split blood specimens; (b) preparing a sample for blood
cell counting by adding a reagent for the blood cell counting to
another of the at least two split blood specimens; (c) irradiating
the sample for the immunoassay and detecting optical information
from a particle in the sample for the immunoassay; (d) irradiating
the sample for blood cell counting and detecting optical
information from a particle in the sample for blood cell counting;
(e) detecting a substance to be immunoassayed based on optical
information from the particle in the sample for the immunoassay;
and (f) performing blood cell counting based on the optical
information from the particle in the sample for blood cell
counting.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a scheme illustrating a configuration of a first blood
analyzer embodying features of the present invention.
FIG. 2 is a plot illustrating carrier particles and blood cells
that emerged at different locations on a two dimensional
scattergram.
FIG. 3 is a histogram of particles that emerged in an area of the
carrier particles on the two dimensional scattergram.
FIG. 4 is a plot illustrating first carrier particles and second
carrier particles that emerged at different locations on the two
dimensional scattergram.
FIG. 5 is a perspective view of a light detecting portion of a
blood analyzer embodying features of the present invention.
FIG. 6 is a plot showing an example of a two dimensional
scattergram.
FIG. 7 is a histogram representing particle distribution of
particles that emerged in an area of the carrier particles on the
two dimensional scattergram.
FIG. 8 is a plot showing an example of a two dimensional
scattergram.
FIG. 9 is a plot showing an example of a two dimensional
scattergram.
FIG. 10 is a scheme illustrating a second blood analyzer embodying
features of the present invention.
FIG. 11 is a plot showing an example of the two dimensional
scattergram.
FIG. 12 is a plot showing an example of the two dimensional
scattergram.
FIG. 13 is a plot showing an example of the two dimensional
scattergram.
DETAILED DESCRIPTION
Blood analyzers embodying features of the present invention and
methods for their use in assays are described below. In blood
analyzers embodying features of the present invention, blood cell
counting and immunoassay by the particle agglutination method are
simultaneously performed for an identical sample. Using the flow
cytometry method, forward scattered light and side fluorescence are
detected as optical information reflecting characteristics of the
respective particles from the sample containing blood cells and
carrier particles. A two dimensional scattergram is made using this
optical information as parameters. Plots corresponding to
respective particles that emerged on the two dimensional
scattergram are differentiated by type of blood cell and carrier
particles depending on their emergence locations, and counted. For
the carrier particles, a degree of agglutination is obtained based
on the optical information detected.
FIG. 1 is a figure showing a configuration of a blood analyzer
embodying features of the present invention. The blood analyzer 1
includes a sample preparing portion 11, a light detecting portion
12, an analyzing portion 13 and an output portion 14.
The sample preparing portion 11 is for preparing an assay sample by
adding given reagents (e.g., carrier particles, a diluent, a
staining solution) to a specimen and reacting them therewith. In
the sample preparing portion 11, the blood cells in the specimen
are fluorescently stained. Carrier particles sensitized with an
antibody or an antigen corresponding to a substance to be
immunoassayed are added in order to perform a particle
agglutination reaction. As carrier particles, it is possible to use
particles typically used for the particle agglutination method, for
example, latex particles, magnetic particles, glass particles,
dendrimers, and the like, and combinations thereof. As the antigen
or antibody which sensitizes the carrier particles when the
substance to be immunoassayed is an antibody, an antigen which
performs an antigen antibody reaction specific for the antibody
maybe used. When the substance to be immunoassayed is an antigen,
then an antibody which performs an antigen antibody reaction
specific to the antigen may be used. For example, when the assay
parameter is CEA antigen (carcinoembryonic antigen), anti-CEA
antibody is sensitized. When the assay parameter is anti-HBs
antibody, HBs antigen is sensitized.
The assay sample prepared at the sample preparing portion 11 is
delivered in solution to the light detecting portion 12. The light
detecting portion 12 is for detecting side fluorescence and forward
scattered light from the particles in the sample by flow cytometry.
The assay sample prepared at the sample preparing portion 11 is
flowed into a flow cell 12a of the light detecting portion 12 and
forms a sample flow. A laser beam is emitted from a laser beam
source 12b, and the sample flow at the flowcell 12a is irradiated.
Then, the side fluorescence generated when the particle in the
sample flow cuts across a laser beam emitted area is received by a
photo multiplier tube 12c and photoelectrically transferred to
electric signals. The forward scattered light generated when the
particle in the sample flow cuts across a laser beam emitted area
is received by a photo diode 12d and photoelectrically transferred
to electric signals.
The electric signals of the side fluorescence and the forward
scattered light detected by the light detecting portion 12 are
delivered to the analyzing portion 13. The analyzing portion 13
includes a computer made up of a hard disc, CPU, ROM, RAM, and on
the like. At the analyzing portion 13, side fluorescence intensity
and forward scattered light intensity are obtained by signals of
each particle. Then, a two dimensional scattergram is made using
the side fluorescence intensity and the forward scattered light
intensity as parameters. The particles that emerged on the two
dimensional scattergram are differentiated into various blood cells
and carrier particles depending on their emergence locations, and
counted.
Since the forward scattered light intensity reflects the size of a
particle, the particles can be differentiated using only the
forward scattered light intensity when the sizes differ by particle
type. Among blood cells (e.g., platelets, erythrocytes and
leukocytes), the platelet is the smallest and the leukocyte is the
largest. However, the sizes of these blood cells are not definitely
defined, and since the sizes overlap for different types of cells,
it is difficult to precisely differentiate based only on size
information of the forward scattered light intensity. Thus, the
side fluorescence intensity is detected as the optical information
reflecting characteristics other than the size of each particle.
When fluorescent staining was previously achieved with a dye that
stains nucleic acid contained in the blood cell, the side
fluorescence intensity detected from the blood cell becomes the
information that reflects amount of the nucleic acid in the blood
cell. The blood cells can be differentiated more precisely by
combining the forward scattered light intensity (size information)
and the side fluorescence intensity (nucleic acid amount
information).
To simultaneously perform blood cell counting and immunoassay, the
carrier particles used for the immunoassay are such that the
emergence locations of the particles do not overlap with those of
the blood cells on the two dimensional scattergram. For example,
the forward scattered light intensity is controlled by altering the
sizes of the carrier particles. In addition, the side fluorescence
intensity is controlled by including a fluorescent dye in the
carrier particle or by altering a concentration of the fluorescent
dye. Thus, if the carrier particles emerge at locations different
from those of the blood cells on the two dimensional scattergram,
it becomes possible to differentiate the blood cells and the
carrier particles. When particles that are much smaller or larger
than the blood cells are used as the carrier particles, it is
possible to differentiate the particles in the sample into carrier
particles and blood cells by difference in forward scattered light
intensity. When carrier particles that are similar in size than the
blood cells are used, the carrier particles and the blood cells can
be differentiated by combining the forward scattered light
intensity and the fluorescence intensity.
For carrier particles differentiated from blood cells on the two
dimensional scattergram, the degree of agglutination is obtained
and the substance to be immunoassayed is detected. The rate of
agglutination described in JP-B-6-19349 is used as the degree of
agglutination based on the forward scattered light intensity of the
carrier particles. This rate of agglutination is calculated as
follows. First, the scattered light intensity of each particle is
obtained by flow cytometry. The particles are then differentiated
into non-agglutinated single particles and agglutinated particles
formed by agglutinating multiple carrier particles according to the
respective scatter intensities. A single particle number (M) and an
agglutinated particle number (P) are counted, a total particle
number (T) which is a sum of M and P is obtained, and P/T is
calculated as the rate of agglutination.
The rate of agglutination of the carrier particles is converted
into a concentration of the substance to be immunoassayed based on
a standard curve previously prepared by measuring specimens
containing the substance to be immunoassayed at known
concentrations. Thus, the concentration of the substance to be
immunoassayed in the unknown specimens is obtained.
An example of a two dimensional scattergram from an assay with a
blood analyzer embodying features of the present invention is shown
in FIG. 2. This figure is a two dimensional scattergram made on the
basis of the forward scattered light and the side fluorescence
detected from a sample prepared by mixing fluorescent latex
particles containing a fluorescent dye as the carrier particles and
a given fluorescent dye for staining the blood cells with a whole
blood specimen. The vertical and horizontal axes represent the
forward scattered light intensity and the side fluorescence
intensity, respectively. The carrier particles emerge segregated
into separate populations because the forward scattered light
intensity differs depending on patterns of agglutination, such as
unagglutinated single particles 21, agglutinated double particles
which occur by agglutinating two carrier particles 22, and
agglutinated triple particles 23 which occur by agglutinating three
carrier particles. As the blood cells, platelets 24 and
erythrocytes 25 emerge, they may be differentiated from the carrier
particles by the difference in forward scattered light intensity
and side fluorescence intensity.
In FIG. 2, G1 is determined beforehand as the area where the
carrier particles emerge. An example of particle size distribution
of the carrier particles which emerge in the area G1 is shown in
the histogram of FIG. 3. The vertical and horizontal axes represent
the particle number (frequency) and the forward scattered light
intensity, respectively. The agglutinated particles and the single
particles are differentiated by determining a threshold for the
forward scattered light intensity.
An antigen (antibody) concentration of each immunoassay parameter
can be obtained by deriving the total particle number T from the
particle number M of the differentiated single particles and the
particle number P of the agglutinated particles (P is the sum of
agglutinated particles equal to or greater than 2), calculating the
rate of agglutination P/T, and performing the concentration
conversion based on the standard curve previously prepared.
The blood cells emerge on the two dimensional scattergram making
populations according to the difference in forward scattered light
intensity and side fluorescence intensity. Thus, the blood cells
are counted by previously determining the area specific for each
type of blood cell and counting the particle number in each area.
In FIG. 2, the areas G2 and G3 are for differentiating the
platelets and the erythrocytes, respectively.
If the emergence locations of the carrier particles and the blood
cells are made different on the two dimensional scattergram by
appropriately controlling the particle diameters of the carrier
particles and the fluorescent dye concentrations contained in the
carrier particles or the concentrations of the fluorescent dye for
staining the blood cells, then the blood cells and the carrier
particles can be definitely differentiated. In addition, when the
immunoassay is carried out with multiple parameters, the first
carrier particles sensitized with the antibody or antigen
corresponding to the first assay parameter and the second carrier
particles sensitized with the antibody or antigen corresponding to
the second assay parameter can be prepared. At that time, the first
and second particles can be differentiated on the two dimensional
scattergram by altering the concentrations of the fluorescent dyes
contained in the first and second carrier particles,
respectively.
FIG. 4 shows an example of the two dimensional scattergram when the
immunoassays are simultaneously carried out for two parameters. The
second carrier particles emerge containing a higher concentration
of the fluorescent dye than the carrier particles that emerge at
the area G1 (first particles). As with the first carrier particles,
the second carrier particles emerge segregated into separate
populations depending on the pattern of agglutination, such as
unagglutinated single particles 31, agglutinated double particles
which occur by agglutinating two carrier particles 32, and
agglutinated triple particles 33 which occur by agglutinating three
carrier particles. The immunoassay can be carried out for the first
and second parameters, respectively, by obtaining the rate of
agglutination from the particle size distribution in each area, for
example, in the area G1 including the first carrier particles and
the area G4 including the second carrier particles.
A display unit such as a CRT, an LCD, and a printer are included at
the output portion 14. The results of the immunoassay and the
various blood cell counts calculated at the analyzing portion 13,
and the two dimensional scattergrams and histograms made upon
analysis are output at the output portion 14.
Hereinafter, experiments are illustrated in which blood cell
counting and immunoassay (e.g., detection of HBs antigen in whole
blood) are carried out by detecting the forward scattered light
intensity and the side fluorescence intensity from samples
containing blood cells and carrier particles and then analyzing the
optical information detected.
For the experiments, a dilution series of seven specimens #1 to #7
containing varying concentrations of HBs antigen were prepared and
added to human normal whole blood. The concentrations of specimens
#1 to #7 are, respectively: 0 U/mL (#1), 1 U/mL (#2), 3 U/mL (#3),
9 U/mL (#4), 27 U/mL (#5), 81 U/mL (#6), and 243 U/mL (#7).
The blood analyzer embodying features of the present invention
shown in FIG. 1 was used for the assay. The blood analyzer 1
includes the sample preparing portion 11, the light detecting
portion 12, the analyzing portion 13, and the output portion 14.
The sample preparing portion 11 includes a specimen supplying
portion 11a, a latex reagent supplying portion 11b, a buffer
supplying portion 11c, a diluent supplying portion 11d, a staining
solution supplying portion 11e, a first reaction vessel 11f, and a
second reaction vessel 11g.
An operator of the blood analyzer 1 sets a latex reagent at the
latex reagent supplying portion 11b prior to operating the
analyzer. The latex reagent is a reagent containing latex particles
of which the surface is sensitized with an antibody or antigen (in
this case, anti-HBs antibody), which performs an antigen antibody
reaction specific to a substance to be immunoassayed. This latex
particle acts as the carrier particle.
The following examples and representative procedures illustrate
features in accordance with the present invention, and are provided
solely by way of illustration. They are not intended to limit the
scope of the appended claims or their equivalents.
Latex Particle Preparation Method
A fluorescent latex particle with particle diameter of 0.78 .mu.m
was used as the carrier particle. The surface of the carrier
particle is sulfate and contains 1% (w/v) red fluorescent dye
(capable of being excited with a laser beam with a wavelength of
633 nm). First, 50 .mu.l of 10% fluorescent latex particle
suspension (w/v) was added to 950 .mu.l of a GTA buffer (0.53 mg/mL
of 3,3-dimethyl glutaric acid, 0.4 mg/mL of Tris, 0.35 mg/mL of
2-amino-2-methyl-1,3-propanediol, pH 4.6) containing 60 .mu.g of an
anti-HBs antibody (mouse monoclonal antibody, commercially
available article) , and left at 20.degree. C. for 2 hours. This
was centrifuged at 10000.times.g for 10 min and a supernatant was
discarded. One (1) mL of a GTA buffer containing 1% (w/v) bovine
serum albumin (commercially available) was added to a pellet and
sonicated to disperse. The procedure from centrifugation through
dispersion was repeated several times. Finally, after centrifuging
and discarding the supernatant, 1 mL of a GTA buffer (pH 6.2)
containing 220 mg/mL of glycerine and 0.3% (w/v) bovine serum
albumin was added to the pellet, and sonicated to disperse, thus
providing the latex reagent.
The operator of the blood analyzer 1 sets the reaction buffer
prepared as follows at the buffer supplying portion 11c prior to
operating the analyzer.
Reaction Buffer Preparation Method
1.6 mg/mL of 3,3-dimethyl glutaric acid, 1.1 mg/mL of
2-amino-2-methyl-1, 3-propanediol, 18.18 mg/mL of Tris, 5% (w/v) of
bovine serum albumin, and 0.8% (w/v) dextran (commercially
available article), pH 6.70, were prepared to provide the reaction
buffer for making the assay sample by adding to the specimen.
The operator of the blood analyzer 1 sets RET SEARCH (II) diluent
(supplied by Sysmex Corporation) at the diluent supplying portion
11d prior to operating the analyzer. This is for diluting the
sample upon staining the blood cells with a staining solution, as
described below.
In addition, the operator of the blood analyzer 1 sets RET SEARCH
(II) staining solution (supplied by Sysmex Corporation) at the
staining solution supplying portion 11e. The RET SEARCH (II)
staining solution contains polymethine fluorescent dye capable of
staining nucleic acid in the blood cells and being excited with a
laser beam at a wavelength of 633 nm. The nucleic acid in the blood
cells is fluorescently stained with this dye.
When the operator of the blood analyzer 1 sets a specimen at the
specimen supplying portion 11a and puts the blood analyzer 1 into
operation, the specimen supplying portion 11a first measures 20
.mu.l of the specimen and delivers it to the first reaction vessel
11f. Next, the buffer supplying portion 11c delivers 160 .mu.l of
the reaction buffer to the first reaction vessel 11f, where the
specimen and the reaction buffer are blended for 15 seconds.
Subsequently, the latex reagent supplying portion 11b delivers 20
.mu.l of the latex reagent to the first reaction vessel 11f, where
the specimen, the reaction buffer, and the latex reagent are
blended and incubated at 45.degree. C. for 15 min to make a latex
reagent mixture sample. Subsequently, 4.5 .mu.l of the latex
reagent mixture sample is delivered from the first reaction vessel
11f to the second reaction vessel 11g. Also, the diluent supplying
portion 11d delivers 0.8955 mL of the RET SEARCH (II) diluent to
the second reaction vessel 11g, where the latex reagent mixture
sample is diluted. Then the staining solution supplying portion 11e
delivers 18 .mu.l of the RET SEARCH (II) staining solution to the
second reaction vessel 11g, where the staining reaction is carried
out for about 31 seconds to prepare the assay sample.
Then, 2.8 .mu.l of the assay sample prepared in this way is
delivered to the light detecting portion 12, and the forward
scattered light and side fluorescence are obtained as optical
information from each particle contained in the sample. A detailed
configuration of the light detecting portion 12 is shown in FIG. 5.
The assay sample given pretreatment such as fluorescent staining is
flowed into the flow cell 41 (cf., 12a in FIG. 1) to form a sample
flow. The laser beam emitted from a semiconductor laser beam source
42 (cf. 12b in FIG. 1) to the sample flow in the flow cell 41
reaches the flow cell 41 through a collimator lens 43, and is
emitted to the sample flow. The forward scattered light which
occurs when the particle in the sample flow cuts across the laser
beam enters the photo diode 46 (cf. 12d in FIG. 1) through a
condenser lens 44a and a pin hole 45a. The side fluorescence enters
the photo multiplier tube 48 (cf. 12c in FIG. 1) through a
condenser lens 44b, a filter 49 and a pin hole 45b. The forward
scattered light signal photoelectrically transferred and output at
the photo diode 46 and the side fluorescence signal
photoelectrically transferred and output at the photo multiplier
tube 48 are sent to the analyzing portion 13.
In the analyzing portion 13, a forward scattered light intensity
and a side fluorescence intensity from particle to particle are
obtained from the forward scattered light signal and the side
fluorescence signal detected at the light detecting portion 12, and
a two dimensional scattergram is made using these as parameters.
FIG. 6 is the two dimensional scattergram obtained by measuring
specimen #6. The vertical and horizontal axes represent the forward
scattered light intensity and the side fluorescence intensity,
respectively. The platelets, the erythrocytes and the carrier
particles form separate populations depending on the differences of
the forward scattered light intensity and the side fluorescence
intensity. In the two dimensional scattergram in FIG. 6, the area
G5 in which the carrier particles are considered to emerge is
determined. Similarly, the areas G6 and G7 are determined in which
the platelets and the erythrocytes, respectively, are considered to
emerge. The determination of the latter area involves the emergence
of reticulocytes, which are larger in fluorescent intensity than
normal erythrocytes. The emergence area of the erythrocytes G7
includes a location of larger fluorescent intensity than that of
mature erythrocytes (i.e., the emergence area of the
reticulocytes).
In FIG. 6, it is found that the carrier particles emerge in the
area G5 segregating into single particles, agglutinated double
particles, and agglutinated triple particles. The analyzing portion
13 makes a histogram showing the particle size distribution of the
particles which emerge in the area G5. FIG. 7 is a histogram
showing the particle size distribution of the particles which
emerge in the area G5. The vertical and horizontal axes represent
the frequency (particle numbers) and the forward scattered light
intensity, respectively. The analyzing portion 13 obtains the total
particle number T from the single particle number M and the
agglutinated particle number P (sum of two or more agglutinated
particles) based on the particle size distribution in the area G5
to calculate the rate of agglutination (P/T %). The rates of
agglutination of the particles obtained by measuring the specimens
#1 to #7 are shown in Table 1.
TABLE-US-00001 TABLE 1 Degree of agglutination Specimen (P/T %) #1
0.37 #2 0.78 #3 1.80 #4 4.91 #5 13.46 #6 29.68 #7 45.31
From Table 1 above, it is found that the rates of agglutination
vary depending on the anti-HBs antibody concentrations contained in
the specimens. Thus, the concentration of the substance to be
immunoassayed in a specimen may be obtained by concentration
conversion of the rate of agglutination on the basis of a standard
curve previously made by measuring the specimens which contain the
substance to be immunoassayed at known concentrations.
FIG. 8 shows a two dimensional scattergram obtained by measuring an
assay sample prepared using the same human normal whole blood as
specimen #1 without adding the latex. The vertical and horizontal
axes represent the forward scattered light intensity and the side
fluorescence intensity, respectively. Since no latex reagent is
contained in the assay sample, no carrier particle emerges on the
two dimensional scattergram in FIG. 8 as compared to FIG. 6. There
is no difference in the emergence locations for the platelets and
the erythrocytes. Thus, it is shown that the presence or absence of
the latex reagent does not influence the results of blood cell
counting.
As described above, on the two dimensional scattergram in FIG. 6,
the areas G6 and G7--where the platelets and the erythrocytes,
respectively, are considered to emerge--are determined beforehand,
and the number of particles which emerge in each area is counted.
Among various blood cells, leukocytes are larger in forward
scattered light intensity and fluorescence intensity compared to
platelets and erythrocytes, and emerge in the location which is not
displayed on the two dimensional scattergram shown in FIG. 6 and
FIG. 8. Thus, the analyzing portion 13 makes the two dimensional
scattergram capable of reflecting the larger forward scattered
light intensity and side fluorescence intensity and differentiating
the leukocytes from the other particles. Then, the area where the
leukocytes are considered to emerge is determined, and the number
of particles in the area is counted. The two dimensional
scattergram for differentiating and counting the leukocytes is
shown in FIG. 9. As with FIG. 6 and FIG. 8, the vertical and
horizontal axes represent the forward scattered light intensity and
the side fluorescence intensity, but the emergence of leukocytes is
identified by extending the display range of the respective
parameters. The populations of platelets, erythrocytes, and carrier
particles that emerged clearly separate on the two dimensional
scattergrams in FIG. 6 and FIG. 8 emerge together at the left
bottom and are not displayed as clearly separate on the two
dimensional scattergram in FIG. 9. The area G8 is the area where
the leukocytes are considered to emerge, and the particles which
emerge in this area are subject to counting as leukocytes.
The analyzing portion 13 counts the particles which emerge in the
areas G6, G7 and G8 on the two dimensional scattergram to make the
platelet number, the erythrocyte number and the leukocyte number,
respectively. Also, the volumes of particles counted as
erythrocytes are calculated based on forward scattered light
intensity and totaled. Then, MCV (mean red cell volume) is
calculated by dividing the total value by the particle number.
The specimen #6 was measured by a blood analyzer embodying features
of the present invention, and various blood cells were
differentiated and counted based on the two dimensional
scattergrams in FIG. 6 and FIG. 9. The results were compared with
the results in which the same whole blood specimen as in specimen
#6 was measured by the conventional method. The conventional method
is one in which the entire operation from setting of the whole
blood specimen to analysis are performed according to the standard
measurement method with an automated hematology analyzer XE-2100
(supplied by Sysmex Corporation). The XE-2100 has a so-called
electric resistance detector in addition to an optical detecting
system, and the platelet number, the erythrocyte number and MCV
(mean red cell volume) are calculated based on the results detected
by the electric resistance detector. The leukocyte number is
calculated based on the results detected by the optical detecting
system.
The measurement results obtained with a blood analyzer embodying
features of the present invention and with the conventional method
are shown in Table 2 below.
TABLE-US-00002 TABLE 2 Present Invention Conventional Method
Erythrocytes 4.70 (10.sup.6 cells/.mu.L) 4.60 (10.sup.6
cells/.mu.L) Platelets 301 (10.sup.3 cells/.mu.L) 302 (10.sup.3
cells/.mu.L) Leukocytes 12.10 (10.sup.3 cells/.mu.L) 12.17
(10.sup.3 cells/.mu.L) MCV 95.8 (fL) 95.8 (fL)
As shown in Table 2 above, a good correlation is obtained for
respective parameters between the blood cell counting results
according to the present invention and the results of the
conventional method.
The analyzing portion 13 has a function which performs a hematocrit
correction for the results of immunoassay of the whole blood
sample. Hereinafter, the hematocrit correction is described.
When a substance to be immunoassayed (e.g., an antibody or an
antigen) is present only in serum/plasma, there is a difference in
concentration of the substance to be immunoassayed for the case
when the immunoassay is performed in the whole blood and the case
when the assay is performed in serum or plasma. This difference is
due to a difference in volume ratio of the blood cell component
that occupies the whole blood (hematocrit value). Since there is an
individual difference in hematocrit values, it is difficult to
precisely correct the assay value in the case when the assay is
performed using the serum or plasma by the method in which the
result of the whole blood immunoassay is multiplied by a constant
coefficient. Thus, if the so-called hematocrit correction is used
when the result of a whole blood immunoassay measured from serum or
plasma is corrected, by the use of the hematocrit value obtained
from the blood cell counting, the same measurement value as that of
the measurement carried out in the serum (or plasma) is
obtained.
The hematocrit value is the volume ratio of the blood cell
component that occupies the whole blood. An example in which a
volume ratio of erythrocytes which occupy a majority of the blood
cell component is used as the hematocrit value is described below.
In the present analyzer, as above, the volume of each particle
counted as the erythrocyte is calculated based on the forward
scattered light signal. The volumes of respective particles are
totaled, and the MCV is calculated by dividing the sum by the
particle number. At the analyzing portion 13, the hematocrit value
is calculated by further multiplying this MCV by the particle
number. When the concentration of the substance to be immunoassayed
in the whole blood is A, the concentration of the substance to be
immunoassayed in the serum (or plasma) is B, and the hematocrit
value (%) is C, then the concentration B of the substance to be
immunoassayed in the serum (or plasma) is obtained from the
following formula: B=A.times.100/(100-C) Using this formula, , the
concentration at the analyzing portion 13 of the substance to be
immunoassayed in the whole blood is converted to the concentration
of the substance to be immunoassayed in the serum (or plasma) by
performing the correction for a result of the immunoassay in the
whole blood using the hematocrit value obtained in the blood cell
counting.
At the output portion 14, the results of the immunoassay as well as
the calculated results of the hematocrit correction, the various
blood cell counts, the two dimensional scattergrams, and the
histograms made upon analysis at the analyzing portion 13 are
provided.
FIG. 10 shows an alternative blood analyzer embodying features of
the present invention. In this example, the sample preparing
portion 11 of the blood analyzer 1 shown in FIG. 1 is made into a
configuration to prepare the sample for the immunoassay and the
sample for the blood cell counting separately. The same reference
numerals are used as in FIG. 1 when the configurations are
common.
Hereinafter, the configuration and performance of the blood
analyzer 1 shown in FIG. 10 is described. The sample preparing
portion 11 includes a specimen supplying portion 11a, a latex
reagent supplying portion 11b, a buffer supplying portion 11c, a
diluent supplying portion 11d, a staining solution supplying
portion 11e, a first reaction vessel 11f, and a second reaction
vessel 11g. Prior to operating the blood analyzer 1, an operator
sets a specimen at the specimen supplying portion 11a, a reaction
buffer at the buffer supplying portion 11c, a diluent at the
diluent supplying portion 11d, and a staining solution at the
staining solution supplying portion 11e. The specimen, latex
reagent, reaction buffer, diluent, and staining solution used are
the same as those in the blood analyzer described above.
When the blood analyzer 1 is started, the specimen supplying
portion 11a first measures 20 .mu.l of the specimen and delivers it
to the first reaction vessel 11f. Next, the buffer supplying
portion 11c delivers 160 .mu.l of the reaction buffer to the first
reaction vessel 11f, where the specimen and the reaction buffer are
blended for 15 seconds. Subsequently, the latex reagent supplying
portion 11b delivers 20 .mu.l of the latex reagent to the first
reaction vessel 11f, where the specimen/reaction buffer and the
latex reagent are blended and incubated at 45.degree. C. for 15 min
to make a sample for the immunoassay.
Subsequently, the specimen supplying portion 11a measures 20 .mu.L
of the specimen and delivers it to the second reaction vessel 11g.
The diluent supplying portion 11d delivers 0.8955 mL of the RET
SEARCH (II) diluent to the second reaction vessel 11g to dilute the
specimen. Next, the staining solution supplying portion 11e
delivers 18 .mu.l of the RET SEARCH (II) staining solution to the
second reaction vessel 11g, where the staining reaction is carried
out for about 31 seconds to prepare a sample for the blood cell
counting.
Subsequently, the sample for immunoassay in the first reaction
vessel 11f is delivered to a flow cell 12a of a light detecting
portion 12, and the side fluorescence signal and the forward
scattered light signal are detected from each particle in the
sample. Next, the sample for blood cell counting in the second
reaction vessel 11g is delivered to the flow cell 12a of the light
detecting portion 12, and the side fluorescence signal and the
forward scattered light signal are detected from each particle in
the sample. The detected signals are sent to an analyzing portion
13. Thus, the sample for immunoassay and the sample for blood cell
counting are delivered to the same flow cells, respectively, and
their optical information is detected. The performance of the light
detecting portion 12 upon the detection of optical information from
respective samples is analogous to the case of the blood analyzer
described in FIG. 1 above.
The analyzing portion 13 makes a two dimensional scatter gram based
on the side fluorescence signal and the forward scattered light
signal detected from the sample for immunoassay. An example of the
two dimensional scattergram is shown in FIG. 11. Since the staining
solution for fluorescently staining the blood cells is not added to
this sample for immunoassay, only carrier particles have strong
fluorescent intensity among the particles involved in the sample.
Thus, S/N ratio is improved when the carrier particles are
differentiated from the particles involved in the sample.
Differentiating of the carrier particles and the calculation of
rate of agglutination are carried out analogous to the case of the
blood analyzer described in reference to FIG. 1 above. Then, the
concentration of the substance to be immunoassayed is obtained by
concentration conversion on the basis of a standard curve.
Also, the analyzing portion 13 makes the two dimensional
scattergram based on the side fluorescence signal and the forward
scattered light signal detected from the sample for blood cell
counting. Examples of the two dimensional scattergrams obtained
from the sample for blood cell counting are shown in FIG. 12 and
FIG. 13. FIG. 12 and FIG. 13 correspond to FIG. 6 and FIG. 9,
respectively. Since the carrier particles for the immunoassay are
not added to this sample for blood cell counting, only blood cells
emerge on the two dimensional scattergram. Then, as is the case
with the blood analyzer described in reference to FIG. 1 above, the
blood cells are differentiated into erythrocytes, leukocytes and
platelets based on the areas determined beforehand on the two
dimensional scattergram, and then counted.
The results of immunoassay and blood cell counting in the analyzing
portion 13 are output at the output portion 14. As is the case with
the blood analyzer shown in FIG. 1, the hematocrit value may be
calculated based on the result of blood cell counting. In
alternative embodiments, the result of immunoassay may be corrected
based on that hematocrit value.
As described above, even if the sample for immunoassay and the
sample for blood cell counting are separately prepared and the
detection of optical information at the light detecting portion 12
is separately performed, the light detecting portion 12 can be
utilized in common with the immunoassay and blood cell
counting.
In the presently preferred embodiments of the invention described
above, the forward scattered light and the side fluorescence were
used as the optical information detected from the samples for blood
cell counting and immunoassay. However, the optical information is
not limited thereto, and those commonly obtained from the blood
cells and carrier particles can be selected from optical
information such as side fluorescence, absorbance, phosphorescence,
chemiluminescence, and bioluminescence. Also, various dyes or
luminescent substrates can be contained in the carrier particles
depending on the optical information used.
Physical properties other than optical information that reflect
characteristics of the particles may be detected from each particle
and differentiating of respective particles may be carried out
based on the physical property. For example, using a physical
property wherein electrodes are disposed to sandwich an orifice as
a detector in a state of applied voltage between the electrodes, a
particle to be assayed is passed through the orifice. When the
particle passes through the orifice, electric resistance takes
place in proportion to the particle size. Thus, the level of this
electric resistance can be detected as the physical property which
each particle has. As the detector for measuring the particle by
utilizing the electric resistance in this way, it is possible to
use, for example, those described in U.S. Pat. No. 5,905,214.
When differentiating of the cells is performed based on the levels
of electric resistance detected from respective particles, it is
preferred that the particle diameter and electric resistance of the
carrier particles are controlled so as to discriminate the blood
cells and carrier particles.
In accordance with the present invention, it becomes possible for
the blood cell counting and immunoassay to be carried out using the
identical measurement section (the light detecting portion in the
embodiment described above). In addition, the specimens and
reagents may be commonly used over multiple assay parameters.
Furthermore, in the immunoassay, the present invention enables
whole blood assay without the need for centrifugation and can
shorten the time period from specimen collection to the obtainment
of the test result. Since the hematocrit correction performed for
the results of immunoassay can be carried out based on blood cell
counting obtained from the same blood sample, the hematocrit
correction can be carried out more precisely.
The foregoing detailed description and accompanying drawings have
been provided by way of explanation and illustration, and are not
intended to limit the scope of the appended claims. Many variations
in the presently preferred embodiments illustrated herein will be
obvious to one of ordinary skill in the art, and remain within the
scope of the appended claims and their equivalents.
* * * * *